Rabbit reticulocyte lysate contains a multiprotein system that assembles steroid receptors into a heterocomplex with hsp90. In the case of the glucocorticoid receptor (GR), the receptor must be bound to hsp90 to bind steroid, and assembly of the GR.hsp90 complex restores the hormone binding domain of the receptor to the steroid binding conformation. Using both direct assay of heterocomplex assembly by Western blotting and indirect assay of assembly by steroid binding, it has previously been determined that the assembly system is both ATP/Mg2+-dependent and K+-dependent and that hsp70 and an acidic 23-kDa protein (p23) are required to form a functional GR.hsp90 complex. It is also thought that a 60-kDa protein (p60) may be required for progesterone receptor.hsp90 heterocomplex assembly, but a complete heterocomplex assembly system has never been reconstituted from individual components. In this work, we separate the proteins of rabbit reticulocyte lysate into three fractions by DEAE chromatography and then reconstitute the GR.hsp90 heterocomplex assembly system in a manner that requires the presence of each fraction. Fraction A contains most of the hsp70 and all of the p60 in lysate, and elimination of p60 by immunoadsorption inactivates this fraction, with bioactivity being restored by the addition of bacterially expressed human p60. The activity of fraction A is replaced by a combination of highly purified rabbit hsp70 and lysate from p60-expressing bacteria. Fraction B contains hsp90, and its activity is replaced by purified rabbit hsp90. Fraction C contains p23, and its activity is replaced in the recombined system by highly purified bacterially expressed human p23. A minimal GR.hsp90 heterocomplex assembly system was reconstituted with purified rabbit hsp70 and hsp90 and bacterially expressed human p23 and p60. This reports the first reconstitution of this apparently ubiquitous protein folding/heterocomplex assembly system.

The glucocorticoid receptor (GR) is recovered from hormone-free cells in a heterocomplex with the molecular chaperone hsp90, which is required to produce the proper folding state for steroid binding. GR⅐hsp90 heterocomplexes are formed by a multiprotein system that appears to exist in all eukaryotic cells. Recently, we have reconstituted a receptor⅐hsp90 heterocomplex assembly system with purified rabbit hsp90 and hsp70 and bacterially expressed human p23 and p60. We have shown that hsp90, p60, and hsp70 form an hsp90⅐p60⅐hsp70 complex that converts the GR from a non-steroid binding to a steroid binding form ( In the current work, we show that the purified rabbit hsp70 utilized in prior studies is contaminated with a small amount of the rabbit DnaJ homolog hsp40. Elimination of the hsp40 from the purified GR⅐hsp90 assembly system reduces assembly activity, and the activity is restored by addition of the purified yeast DnaJ homolog YDJ-1. hsp40 is a component of the hsp90⅐p60⅐hsp70 foldosome complex isolated from reticulocyte lysate with antibody against p60. Under conditions that promote binding of p23 to hsp90 (elevated temperature, ATP, Nonidet P-40, molybdate), a five-membered (p23⅐hsp90⅐p60⅐hsp70⅐hsp40) complex of chaperone proteins is formed in reticulocyte lysate or from purified proteins. The hsp40-free, purified assembly system has a modest level of assembly activity that is maximally potentiated by YDJ-1 when it is present at about one-twentieth the concentration of hsp70. Although hsp40 is not in the final GR⅐hsp90 heterocomplex isolated from L cell cytosol, it is in the GR⅐hsp90 heterocomplex assembled in reticulocyte lysate. We conclude that hsp40 is a component of the multiprotein hsp90-based chaperone system where it potentiates GR⅐hsp90 heterocomplex assembly.

In cytosols from animal and plant cells, the abundant heat shock protein hsp90 is associated with several proteins that act together to assemble steroid receptors into receptor⅐hsp90 heterocomplexes. We have reconstituted a minimal receptor⅐hsp90 assembly system containing four required components, hsp90, hsp70, p60, and p23 In this work, we show that addition of p23 to native GR⅐hsp90 heterocomplexes immunoadsorbed from L cell cytosol or to GR⅐hsp90 heterocomplexes prepared with the minimal (hsp90⅐p60⅐hsp70) assembly system inhibits both receptor heterocomplex disassembly and loss of steroid binding activity. p23 stabilizes the GR⅐hsp90 heterocomplex in a dynamic and ATP-independent manner. In contrast to hsp90 that is bound to the GR, free hsp90 binds p23 in an ATP-dependent manner, and hsp90 in the hsp90⅐p60⅐hsp70 heterocomplex is in a conformation that does not bind p23 at all. The effect of p23 in the minimal GR heterocomplex assembly system is to stabilize GR⅐hsp90 heterocomplexes once they are formed and it does not appear to affect the rate of heterocomplex assembly. Molybdate has the same ability as p23 to stabilize GR heterocomplexes with mammalian hsp90, but GR heterocomplexes with plant hsp90 are stabilized by p23 and not by molybdate. We propose that incubation of the GR with hsp90⅐p60⅐hsp70 forms a GR⅐hsp90 heterocomplex in which hsp90 is in an ATP-dependent conformation. The ATP-dependent conformation of hsp90 is required for the hormone binding domain to have a steroid binding site, and binding of p23 to that state of hsp90 stabilizes the GR⅐hsp90 heterocomplex to inactivation and disassembly.

Rabbit reticulocyte lysate contains a multiprotein chaperone system that assembles steroid receptors into a complex with hsp90. The glucocorticoid receptor (GR) is bound to hsp90 via its hormone binding domain (HBD), which must be associated with hsp90 to have a steroid binding conformation. Recently, we have reconstituted a receptor⅐hsp90 heterocomplex assembly system with purified rabbit hsp90 and hsp70 and bacterially expressed human p23 and p60 (Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., and Pratt, W. B. (1996) J. Biol. Chem. 271, 12833-12839). In this work we show that when the GR is incubated with hsp90, hsp70, and p60, steroid binding sites are generated despite the absence of p23. In this minimal reconstituted system, the GR is incubated with the chaperones in the presence of

A system consisting of five purified proteins: Hsp90, Hsp70, Hop, Hsp40, and p23, acts as a machinery for assembly of glucocorticoid receptor (GR)⅐Hsp90 heterocomplexes. Hop binds independently to Hsp90 and to Hsp70 to form a Hsp90⅐Hop⅐Hsp70⅐Hsp40 complex that is sufficient to convert the GR to its steroid binding form, and this four-protein complex will form stable GR⅐Hsp90 heterocomplexes if p23 is added to the system (Dittmar, K. D., Banach, M., Galigniana, M. D., and Pratt, W. B. (1998) J. Biol. Chem. 273, 7358 -7366). Hop has been considered essential for the formation of receptor⅐Hsp90 heterocomplexes and GR folding. Here we use Hsp90 and Hsp70 purified free of all traces of Hop and Hsp40 to show that Hop is not required for GR⅐Hsp90 heterocomplex assembly and activation of steroid binding activity. Rather, Hop enhances the rate of the process. We also show that Hsp40 is not essential for GR folding by the five-protein system but enhances a process that occurs less effectively when it is not present. By carrying out assembly in the presence of radiolabeled steroid to bind to the GR as soon as it is converted to the steroid binding state, we show that the folding change is brought about by only two essential components, Hsp90 and Hsp70, and that Hop, Hsp40, and p23 act as nonessential co-chaperones.The steroid receptors are recovered from cells as multiprotein heterocomplexes containing a dimer of Hsp90, substochiometric amounts of Hsp70, 1 an acidic 23-kDa protein, p23, and a tetratricopeptide repeat domain protein, such as immunophilin or protein phosphatase 5 (for review see Refs. 1 and 2). The steroid receptor⅐Hsp90 heterocomplexes can be formed under cell-free conditions by incubating the immunoadsorbed proteins with reticulocyte lysate (3, 4). Inasmuch as the glucocorticoid receptor (GR) 2 must be associated with Hsp90 for it to have steroid binding activity (5), incubation of Hsp90-free GR with reticulocyte lysate results in generation of steroid binding activity in direct proportion to the number of GR⅐Hsp90 heterocomplexes that are assembled (6). Hsp90 binds directly to the ligand-binding domain (LBD) of the GR (1), and complexing with Hsp90 also opens up both thiol moieties (7) and trypsin cleavage sites (8, 9) in the LBD to attack by a thiol-derivatizing agent and the protease. These biochemical observations, coupled with data derived from GR mutants (10), support the idea (6, 11) that the Hsp90 heterocomplex assembly system in reticulocyte lysate directs an ATP-dependent partial unfolding of the GR LBD, thus opening the hydrophobic steroid-binding cleft to access by steroid.The heterocomplex assembly system has been reconstituted (12-16), and five proteins, including Hsp90, Hsp70, Hop (60-kDa Hsp organizer protein), Hsp40, and p23, participate in the ATP/Mg 2ϩ -dependent and K ϩ -dependent assembly process (for review of heterocomplex assembly see Refs. 17 and 18). Besides Hsp90 itself, the only component of this Hsp90-based chaperone system proven to be essential for both GR⅐Hsp90 heterocomplex...

The glucocorticoid receptor (GR) HBD must be bound to the protein chaperone hsp90 in order to acquire the high affinity steroid binding conformation. Despite this crucial role of hsp90, its binding site in GR remains poorly defined. Large portions of the GR HBD have been implicated and no similarity has been established between steroid receptor HBDs and the catalytic domains of the protein kinases (e.g. pp60 src , Raf) that also form stable heterocomplexes with hsp90. Thus, it has been thought that some general property of the proteins, such as exposure of hydrophobic residues in partially denatured regions, determines the assembly of stable hsp90 heterocomplexes. In this work, we have studied fusion proteins containing glutathione S-transferase (GST) and very short amino-terminal truncations just before and at the beginning of the rat GR HBD that are otherwise intact to the carboxyl terminus. Overexpression in COS cells of the chimeras GST537C and GST547C was found to yield receptors that were bound to hsp90 and had wild-type steroid binding affinity. However, removal of 7 more amino acids to form GST554C resulted in a fusion protein that did not bind either hsp90 or steroid. Additional mutations revealed that the role of these 7 amino acids was neither to provide a spacer between protein domains nor to expose a protein surface by introducing a bend in the conserved ␣-helix. Instead, these observations support a model in which the sequence of the 7 amino acids directly or indirectly affects hsp90 binding to the GR HBD. Thus, a region of GR that has not been thought to be relevant for hsp90 binding is now seen to be of critical importance, and these data argue strongly against the commonly accepted model of receptor-hsp90 heterocomplex assembly in which the chaperone initially interacts nonspecifically with hydrophobic regions of the partially denatured HBD and subsequently assists its folding to the steroid binding confirmation. The hormone-binding domain (HBD)1 of the glucocorticoid receptor (GR) is comprised of the carboxyl-terminal one-third of the protein (for review, see Refs. 1 and 2). However, the HBD cannot bind steroid unless the receptor is bound to the chaperone hsp90 (3, 4). hsp90 is a component of a multiprotein chaperone system (including hsp70, p60, and p23) that directs the ATP-dependent assembly of hsp90 into complexes with a variety of transcription factors and protein kinases (for review, see Refs. 5 and 6). It is not known what properties permit these proteins to form relatively stable complexes with hsp90, whereas most proteins do not. It is clear, though, that hsp90 binds directly to the GR HBD (7,8). When the GR HBD is fused to another transcription factor, the fusion protein is bound to hsp90 and binds steroid (9). Furthermore, the activity of the fused transcription factor is usually controlled by steroid binding (for review, see Ref. 10).Several approaches have been taken to determine the region of the GR HBD involved in steroid binding and hsp90 binding. When GR was translated in reti...

The heat shock protein hsp70/hsc70 is a required component of a five-protein (hsp90, hsp70, Hop, hsp40, and p23) minimal chaperone system reconstituted from reticulocyte lysate that forms glucocorticoid receptor (GR)⅐hsp90 heterocomplexes. BAG-1 is a cofactor that binds to the ATPase domain of hsp70/hsc70 and that modulates its chaperone activity. Inasmuch as BAG-1 has been found in association with several members of the steroid receptor family, we have examined the effect of BAG-1 on GR folding and GR⅐hsp90 heterocomplex assembly. BAG-1 was present in reticulocyte lysate at a BAG-1:hsp70/hsc70 molar ratio of ϳ0.03, and its elimination by immunoadsorption did not affect GR folding and GR⅐hsp90 heterocomplex assembly. At low BAG-1:hsp70/ hsc70 ratios, BAG-1 promoted the release of Hop from the hsp90-based chaperone system without inhibiting GR⅐hsp90 heterocomplex assembly. However, at molar ratios approaching stoichiometry with hsp70, BAG-1 produced a concentration-dependent inhibition of GR folding to the steroid-binding form with corresponding inhibition of GR⅐hsp90 heterocomplex assembly by the minimal five-protein chaperone system. Also, there was decreased steroid-binding activity in cells that were transiently or stably transfected with BAG-1. These observations suggest that, at physiological concentrations, BAG-1 modulates assembly by promoting Hop release from the assembly complex; but, at concentrations closer to those in transfected cells and some transformed cell lines, hsp70 is continuously bound by BAG-1, and heterocomplex assembly is blocked.A number of signaling proteins, including several members of the nuclear receptor family, the dioxin receptor, nitric-oxide synthase, and several protein kinases, exist in cytosolic complexes with the ubiquitous and abundant heat shock protein (hsp) 1 hsp90 (for review, see Refs. 1 and 2). These signaling protein⅐hsp90 heterocomplexes can be formed under cell-free conditions by incubating the immunoadsorbed proteins with reticulocyte lysate (3, 4). A minimal heterocomplex assembly system has been reconstituted (5-9); and five proteins, including hsp90, hsp70, 2 Hop (60-kDa hsp organizer protein), hsp40 and p23, participate in the ATP/Mg 2ϩ -dependent and K ϩ -dependent assembly process (for review of heterocomplex assembly, see Refs. 10 and 11). Hip and BAG-1 (Bcl-2-associated gene product-1) are co-chaperones of hsp70 that are potential participants in or regulators of this multiprotein hsp90-based chaperone system. BAG-1 is an hsp70-binding protein that associates with the ATPase domain of the molecular chaperone with a K D of ϳ1-10 nM (12). BAG-1 was originally cloned from a mouse library as a Bcl-2-binding protein and shown to have anti-apoptotic activity (13). Subsequently, BAG-1 was shown to form complexes with some signaling proteins, including receptors for hepatocyte growth factor and platelet-derived growth factor (14), the serine/threonine protein kinase Raf-1 (15), and the retinoic acid receptor (16). An isoform of the human BAG-1 protein called RAP...

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